Studies on the pathogenesis of and propagation of enterovirus 71 in poliomyelitis-like disease in monkeys

Summary Poliomyelitis-like disease in monkeys infected with enterovirus 71 (E71) were studied to determine whether nerve cell damage is due primarily to virus multiplication in the neurons or secondary to the effects of virus multiplication in the supporting tissue of the central nervous system (CNS). Monkeys infected with E71 develop a disease with neuromuscular or muscular dysfunction and lesions of the CNS. E71 was recovered from the lumbar and cervical cord and from the cerebrum. Specific immunofluorescence was detected in the degenerating or necrotic nerve cells in the anterior grey horns of the lumbar and cervical cord, the cerebellum and cerebrum, although virus antigen was not found in the white matter. No specific immunofluorescence was found in the glial cells, vascular endothelial or mononuclear inflammatory cells. From the present results, it is suggested that nerve cell damage is due principally to virus multiplication in the neurons of the CNS in monkeys infected with E71.     

PATHOGENICITY OF A POLIOMYELITIS-LIKE DISEASE IN MONKEYS INFECTED ORALLY WITH ENTEROVIRUS 71: A MODEL FOR HUMAN INFECTION

Hashimoto I. & Hagiwara A. (1982) Neuropathology and Applied Neurobiology 8, 149–156
Pathogenicity of a poliomyelitis-like disease in monkeys infected orally with enterovirus 71: a model for human infection
Ten cynomolgus monkeys were given enterovirus 71 (E71) by mouth. Clinically, only one monkey showed weakness of the lower extremities. Histopathologically, vascular lesions of variable intensity, perivascular cuffing, degeneration and necrosis of the neurons and neuronophagia were observed in the CNS of 7 monkeys. E71 was recovered from the CNS and specific immunofluorescence was detected in the neurons and in associated macrophages in the CNS. Serum neutralizing antibody titres rose from 14 to 21 days. These monkeys are as susceptible to E71 infection by the oral route as by the subcutaneous route as previously shown, and its neuronal virulence was confirmed by its producing CNS lesions after oral infection. The orally infected monkey with E71 appears to provide an excellent model for infection by this agent in man.     

Ultrastructural studies on the pathogenesis of poliomyelitis in monkeys infected with polivirus

Summary Poliovirus was inoculated intraspinally into cynomolgus monkeys to determine whether nerve cell damage in the central nervous system (CNS) is due primarily to virus multiplication in the neuron or to secondary effects of virus multiplication in the supporting cells. Electron-microscopically, the development of cytopathogenesis and of membrane-bound vesicles and virus particles in the neurons of the CNS in monkeys infected with poliovirus was compared with that of infected cultured cynomolgus monkey kidney (CMK) cells. The structure of membrane-bound vesicles in cytoplasm of damaged motoneuron was examined and found to be similar to the vesicles in infected cultured CMK cells. Virus-like particles were detected occasionally around or within membrane-bound vesicles in the cytoplasm of degenerating motoneurons as well as cultured CMK cells, although intracytoplasmic crystals were not detected in the neuron. No virus particles or membrane-bound vesicles were found in astrocyte foot plates, microglia, oligodendrocytes, axons, vascular endothelial, and inflammatory cells. In addition, poliovirus antigen was detected only in the nerve cells of the CNS by the immunoperoxidase technique, although specific staining was never found in the supporting tissues. From the present results we suggest that membrane-bound vesicles in the cytoplasm of the motoneuron are closely correlated with virus multiplication and that damage of the nerve cell is due to the direct action of the poliovirus.     

The distribution of herpes simplex type 1 antigen in mouse central nervous system after different routes of inoculation

Herpes simplex type 1 virus was inoculated into 3-week-old mice via four different routes; intracerebral, intravenous, intranasal and directly into the sciatic nerve. Virus antigen-containing cells in the central nervous system were identified by both an immunofluorescence and immunoperoxidase method. The portal of entry of virus into the CNS appeared to be the major determinant of distribution of virus antigen. Direct haematogenous seeding of virus into the CNS was not proven. It seems probable that infection was first established in sensory ganglia. Within the CNS, regions of high virus antigen concentration paralleled high cell density suggesting cell to cell spread. Consistent involvement of certain neuron groups may be due to their selective vulnerability. These animal experiments provide some explanation for the patterns of CNS herpetic infection observed in man.     

Herpes simplex virus infection and damage in the central nervous system: immune response to myelin basic protein

Two models of herpes simplex virus (HSV) infection in the mouse (inoculation of the ear pinna or intravenous inoculation) were used to assess immune responses to the virus and myelin basic protein (MBP). In both models demyelination in the central nervous system (CNS) can be assessed by clinical signs; ear paralysis (ear inoculation) and hind limb paralysis (intravenous inoculation). In the ear model, low levels of antibody to MBP were detected at 7 and 14 days after inoculation. The incidence of such antibody was low and bore no correlation with signs of paralysis. Cells from draining lymph nodes of mice inoculated in the pinna showed a proliferative response to HSV and MBP in vitro. In both models, delayed-type hypersensitivity reactions were demonstrated against HSV and MBP but such reactions were most significant after i.v. inoculation. This may reflect the greater CNS damage associated with hind limb paralysis.     

空肠弯曲菌黏附蛋白(PEB1)基因工程疫苗外周神经安全性的实验研究

目的 以空肠弯曲菌(campylobacter jejuni，CJ)菌体全抗原为对照，以基因重组的该菌融合黏附蛋白(TrxA／PEB1)作为CJ疫苗免疫大鼠，并对其神经安全性进行实验性评价。方法分别以纯化的TrxA／PEB1和CJ Pen19菌体全抗原经全身免疫增强法免疫大鼠，初次免疫后第2、4、5周采用ELISA法动态观察抗体滴度，并分别于第3、5周分离坐骨神经原纤维及行甲苯胺蓝染色光镜观察。采用ELISA法检测第5周抗血清与GM1抗原的结合反应。神经外膜下注射抗TrxA／PEB1和抗CJ全菌抗原的特异性抗血清，观察其神经病理学改变。结果 (1)经两种抗原免疫后，大鼠均产生高滴度特异性IgG，且各自与两种抗原发生交叉反应I(2)以CJPen19全抗原免疫后抗血清可与GM1抗原结合，而TrxA／I)EBI免疫者则否，(3)CJPenl9免疫组有60％大鼠坐骨神经出现以轴索变性为主的免疫性损伤。而TrxA／PEB]融合蛋白免疫者则未发生；(4)CJ Pen19免疫血清神经外膜下注射后全部大鼠均发生轴索变性为主的病变。且75％为显著病变，而TrxA／PEB1免疫血清外膜下注射后却未引起明显病变。结论CJ Pen19全菌抗原和。TrxA／PEB1纯化蛋白作为免疫原均可激发大鼠有效体液免疫应答，但与CJ全菌抗原相比，TrxA／PEB1蛋白所产生的全身免疫反应未引起周围神经损伤，其抗血清不含针对GMl抗原的抗体，对外周神经元免疫损伤作用。据此可认为TrxA／PEB1作为CJ疫苗安全可靠。     

Anti-myelin associated glycoprotein antibodies recognize HNK-1 epitope on CNS

Antibodies to myelin-associated glycoprotein (MAG) are associated with demyelinating polyneuropathy and are specific for the HNK-1 epitope. To test if anti-MAG IgM recognize HNK-1 on CNS, sera from 20 patients and 238 controls were tested on rat slices by indirect immunofluorescence (IIF). IgM from anti-MAG positive patients, but not from control sera, stained rat brain with perineuronal or neuropil pattern, depending on the CNS region. IIF titers significantly correlated with ELISA anti-MAG titers. The staining of patients' sera were inhibited by mouse anti-HNK-1 monoclonal antibody. Our results demonstrate that anti-MAG IgM recognizes HNK-1 outside the peripheral nerve myelin carriers.     

Experimental panencephalitis induced in suckling mice, by parainfluenza type 1 (6/94) virus, II. Virologic studies.

6/94 virus, a parainfluenza type 1 isolate from multiple sclerosis brain tissue, produced a chronic panencephalitis when inoculated intracerebrally into suckling ICR mice. Immunofluorescent staining revealed 6/94 viral antigen in ependyma, meninges, choroid plexus, and perivascular parenchymal sites from day 3 to 128 days after infection. Hemadsorption-neutralizing antibody was first detected between 20-25 days after infection and remained at high titers for 7 months. Using embryonated chicken eggs, virus was recovered from mouse brains for only 8 days, but could be recovered from brains grown in vitro as explants for 37 days after infection. In cell lines established from explanted brain tissue, immunofluorescence was the most sensitive indicator of virus presence, although infectious virus was not produced. Fusion of these mouse brain cells with human (W138) indicator cells was the most effective means of rescuing 6/94 virus.     

Peripheral,but not central nervous system,type I interferon expression in mice in response to intranasal vesicular stomatitis virus infection

Type I interferon (IFN) is critical for resistance of mice to infection with vesicular stomatitis virus (VSV). Wild type (wt) VSV infection did not induce type I IFN production in vitro or in the central nervous system (CNS) of mice; however IFN-beta was detected in lungs, spleen, and serum within 24 h. The M protein mutant VSV, T1026R1 (also referred to as M51R), induced type I IFN production in vitro and in the CNS, with poor expression in spleens. In addition, VSV T1026R1 was not pathogenic to mice after intranasal infection, illustrating the importance of IFN in controlling VSV replication in the CNS. Experiments with chemical sympathectomy, sRAGE, and neutralizing antibody to VSV were performed to investigate the mechanism(s) utilized for induction of peripheral IFN; neither sRAGE infusion nor chemical sympathectomy had an effect on peripheral IFN production. In contrast, administration of neutralizing antibody (Ab) readily blocked the response. Infectious VSV was transiently present in lungs and spleens at 24 h post infection. The results are consistent with VSV traffic from the olfactory neuroepithelium to peripheral lymphoid organs hematogenously or via lymphatic circulation. These results suggest that VSV replicates to high titers in the brains of mice because of the lack of IFN production in the CNS after intranasal VSV infection. In contrast, replication of VSV in peripheral organs is controlled by the production of large amounts of IFN.     

Changes in sciatic nerve and spinal cord function induced by a CNS viral infection

Electrophysiologic response characteristics of mouse sciatic nerve and spinal cord were investigated following CNS infection with the temperature-sensitive (ts) vesicular stomatitis virus (VSV) mutant G31 KS5. Measurements were obtained before clinical symptoms of the virus-induced CNS disease appeared. Sciatic nerve peak conduction velocities were not different between virus and control inoculated groups. For all control groups, sciatic nerve response (SNR) recovery, characterized by the amplitude ratio of double pulse-evoked responses, followed a facilitation-depression time course. By 4 days after VSV inoculation, the time course of SNR recovery changed with the SNR amplitude ratios significantly depressed compared to control. Crossed spinal responses (CSRs) were measured from one sciatic nerve in response to stimulation of the contralateral sciatic nerve. For all control groups, CSR recovery, as characterized by area ratios calculated from single and double pulse evoked responses, followed a facilitation-depression time course. By 5 days after VSV inoculation, the time course of CSR recovery changed with the CSR area ratios significantly depressed compared to control. The results show that simple electrophysiologic techniques can be used to detect virus-induced changes in sciatic nerve and spinal cord previously undetectable by clinical measures.     

NEUROPATHOLOGY AND NEUROVIRULENCE OF CANINE DISTEMPER VIRUS PLAQUE ISOLATES IN THE HAMSTER

The relationship between neuropathological abnormalities, antibody response and neurovirulence of plaque isolates has been studied in an experimental model of canine distemper in the hamster. Genetic virus variance influenced neurovirulence and the experimental evidence supports the hypothesis that the mechanism of this effect may be through the modulating effect of circulating antibody. Large plaque virus (LPV) produced severe encephalitis with little early antibody response and a high degree of pathological abnormality. Small plaque virus (SPV) produced mild chronic encephalitis and early antibody response. Microscopically, histological abnormalities in this group were qualitatively similar to those seen with LPV but generally of lesser degree. Immunosuppression in SPV infected animals increased the severity of the encephalitis, reflected by the increase in inflammation and inclusion formation. Combined SPV and LPV infection produced high antibody levels and less severe disease than LPV infection alone with an intermediate pattern of histological abnormality.     

Antibody in SSPE serum and brain IgG against measles virus smooth nucleocapsids detected by immunoelectron microscopy

Summary SSPE patients characteristically have high complement-fixing (CF) and neutralizing (N) antibody titers against measles virus in their sera, CSF, and brain. However, using SSPE patients' sera, the immunoperoxidase (IP) labeling of smooth nucleocapsids in SSPE or measles virus infected Vero cells or measles virions has not been achieved using conventional EM fixatives. Using the periodate-lysine-paraformaldehyde (PLP) fixative developed by McLean and Nakane (1974), and the indirect IP technique, antibodies against smooth nucleocapsids in SSPE and measles virus infected Vero cell cultures have been detected in serum from SSPE patients and normal individuals with high measles antibody titers.     

Neurovirulence of two clonally related herpes simplex virus type 1 strains in a rabbit seizure model

Herpes simplex virus type 1 (HSV-1) strains vary widely with regard to neurovirulence, but their tropism for specific central nervous system structures and their ability to induce seizures are poorly defined. We have used the clonally related +GC and -GC strains of HSV-1 to define the pathophysiological basis of neurovirulence in a rabbit model. Following intranasal inoculation, +GC infection was nearly uniformly fatal while -GC infection was asymptomatic. The +GC infected animals developed electroencephalographic (EEG) abnormalities which preceded severe motor seizures. Tropism of the +GC strain for specific CNS nerve centers and the expression of viral antigens within them correlated with its virulence. Although both viruses invaded and replicated within the brain, +GC replicated to slightly higher titers and expressed more abundant viral antigen than -GC. The relatively less efficient replication of -GC appeared to correlate with its temperature-sensitive phenotype in vitro. Both +GC and -GC antigens were found in cerebral cortical layers IV-VI, and in several central nervous system trigeminal and olfactory system structures. However, +GC spread more completely throughout the brain to involve the amygdala, nucleus accumbens, several brainstem nuclei and the locus ceruleus. The +GC antigens were also found in cerebral cortical layer I of animals that developed seizures. These results indicate that the ability of HSV-1 to induce electrophysiologic brain abnormalities is associated with its ability to replicate within specific brain nerve centers.     

Pathogenesis of human poliovirus infection in mice. I. Clinical and pathological studies

Human poliovirus infection in mice was studied to determine the similarities to human poliomyelitis, the selective vulnerability of neurons to infection, the role of the immune response in age-dependent susceptibility, and possible viral persistence. Mice inoculated intracerebrally (ic) with the Lansing type 2 poliovirus developed a disease with clinical, pathological, and age-dependent features resembling human poliomyelitis. Adult mice had a shorter incubation period (50% paralysis, Day 8 vs. Day 13) and a higher incidence of paralysis (97% vs. 79%) than newborns. Only paralyzed animals had pathologic changes in the spinal cord, and these corresponded to the degree of paralysis. Fluorescent antibody staining showed that selective infection of neurons was most intense in the anterior horn motor neurons of the spinal cord. There was no extraneural virus replication and no systemic neutralizing antibody response. Cyclophosphamide immunosuppression enhanced rather than diminished disease, indicating that maturation of immune responses did not explain the relative resistance of newborns to paralysis.     

Inflammation in neuroviral diseases

During any viral infection of the central nervous system (CNS), the extent and nature of neural cell alterations are dictated by the localization of virus replication and, possibly, persistence. However, one additional source of CNS damage comes from the immune response that develops following CNS viral infection. Indeed, despite of its major role in controlling virus spread in the infected CNS, the immune system is equipped with numerous molecular effectors shared with the nervous system that may greatly alter the homeostasis and function of neural cells. Proinflammatory cytokines and metalloproteases belong to this inflammatory cascade. Besides neurovirulence, the crosstalk engaged between neural and immune cells is a major factor determining the outcome of neuroviral infections.     

Replication of the scrapie agent in hamster brain precedes neuronal vacuolation

The scrapie agent causes a degenerative neurological disorder in sheep and goats after a prolonged incubation period. Hamsters inoculated intracerebrally with 10(7) ID50 units of the scrapie agent develop clinical signs of neurological dysfunction 60-65 days later. The titers of scrapie agent in selected regions of the central nervous system (CNS) of hamsters were determined prior to the onset of clinical illness. At 48 days after inoculation, the cerebrum, cerebellum, brain stem, and spinal cord contained 9.3, 9.1, 9.3, and 8.6 log ID50 units/g of tissue, respectively. Sections from the cerebrum showed minimal vacuolation without any astrogliosis. The spinal cord and cerebellum revealed no lesions. At 71 days after inoculation, when clinical signs of scrapie were prominent, another group of hamsters was evaluated. The mean titers of the agent in the same CNS regions were virtually unchanged, but severe vacuolation and moderate astrogliosis were present in the cerebral cortex. A moderate degree of vacuolation and astrogliosis were observed in the cerebellum, brain stem, and spinal cord. These studies indicate that replication of the scrapie agent in the hamster is uniform throughout the CNS and precedes the development of pathological changes.     

In the absence of glycoprotein I (gI), gE determines bovine herpesvirus type 5 neuroinvasiveness and neurovirulence

Bovine herpesvirus type 5 (BHV-5) is an alphaherpesvirus that causes fatal encephalitis in calves. Envelope glycoproteins E (gE) and gI of alphaherpesviruses are important for the pathogenesis in vivo. Previously the authors determined that BHV-5 gE is important for BHV-5 neurovirulence. To determine the role of gI in BHV-5 neurovirulence, the authors have constructed gI-deleted and gI-revertant BHV-5 and analyzed their neuropathogenic properties in a rabbit seizure model. Following intranasal infection, 40% of the rabbits infected with the gI-deleted virus showed severe neurological signs. gI-deleted BHV-5 invaded all the central nervous system (CNS) structures invaded by the gI-revertant BHV-5; however, the number of neurons infected by the gI-deleted virus was similar or slightly reduced (two to four fold). Thus, the gI-deleted virus retained significant neurovirulence and/or neuroinvasive properties when compared with the gE-deleted BHV-5. Pulse-chase analysis revealed that the gE of gI-deleted virus was processed to a larger and a diffused 94- to 100-kDa protein (instead of 94 kDa). The 94- to 100-kDa protein was processed in the Golgi with delayed kinetics but it was endoglycosidase H (EndoH) resistant. In cells infected with gI-deleted virus, there was a reduction in cell-surface gE expression compared to wild-type, which correlated to reduced amount of gE processed in the Golgi. The authors believe that in the absence of gI, BHV-5 gE is sufficient for BHV-5 neurovirulence.     

Mononeuritis des N. oculomotorius bei infektiöser Mononukleose

A 19-year-old immune-competent patient developed right-sided headache and, subsequently, subacute diplopia. On clinical examination he had incomplete right oculomotor palsy. Cranial MRI showed pathologic contrast enhancement of the right oculomotor nerve at its exit point from the mesencephalon, and the CSF displayed slight pleocytosis. The following relevant differential diagnoses were not supported by additional examinations: neurosarcoidosis, Lyme neuroborreliosis, neurosyphilis, tuberculous meningitis, viral meningitis (HIV, VZV, CMV), CNS lymphoma, vasculitis associated with rheumatic disease, Tolosa-Hunt syndrome, and diabetic neuropathy. However, on the basis of blood lymphocytosis, positive heterophile antibody test (Paul-Bunnell test), the presence of IgM antibodies against Epstein-Barr virus capsid antigen, and elevated transaminases, infectious mononucleosis was diagnosed. Isolated neuritis of the oculomotor nerve is a rare parainfectious manifestation of infectious mononucleosis.     

ICAM-5 modulates cytokine/chemokine production in the CNS during the course of herpes simplex virus type 1 infection

Chemokines are important in HSE development in the CNS but underlying regulatory events are unknown. Two-hybrid binding assays identified that intercellular adhesion molecule 5 (ICAM-5), an immune modulator in the CNS, interacted with neurovirulence factor, UOL, of HSV-1. Viral load and interleukin levels were similar in UOL deletion virus (ΔUOL), and wild type virus infected mouse brains. However, higher numbers of lymphocytes, but unaltered soluble ICAM-5 and chemokine levels were detected in ΔUOL infected mouse brains. In contrast, lower lymphocyte numbers, reduced soluble ICAM-5, and higher chemokine levels were detected in wild type virus infected brains. Our results suggest that ICAM-5 plays a critical role in modulating chemokine production in the CNS.